This invention relates to compounds containing a cleavable linker between a deliverable compound, e.g., a pharmaceutical agent, and a targeting moiety, e.g., a protein.
There are many diagnostic and therapeutic agents which suffer from non-selectivity of effect. Thus, cells not in need of a particular treatment are nevertheless exposed to the treating agent. Analogously, cells not intended to be the subject of a diagnosis, e.g., an imaging procedure such as radioimaging or MRI, are nevertheless subjected thereto, i.e., are imaged. As a result of this pervasive non-selectivity problem, much effort has been expended to enhance the selectivity of such pharmaceutical agents.
One technique often used is the binding of the non-selective pharmaceutical agent to another chemical moiety which is capable of targeting the resultant conjugate to a desired site. In most cases, the conjugate must also provide a means for cleaving the "active" agent from the targeting portion. This combination of features poses a unique biochemical problem: Not only must the conjugate provide a targeting moiety specific for a given site, but the cleavability must also be functional at that site and in such a manner that the agent retains its therapeutic or diagnostic capabilities.
In one general method, a protein such as a monoclonal antibody is attached to the pharmaceutical agent. The antibody is selected to be specific for the type of cells desired to be treated or diagnosed. The targeting portion has been attached to the pharmaceutical agent by a variety of linking groups, some of which are cleavable linkers.
Heretofore, the diphosphate (DP) group has been used as a linker between certain molecules. This group is hydrolyzed (cleaved) in situ by enzymes such as phosphodiesterase (EC 3.1.4.1), 5'-nucleotidase (EC 3.1.3.5), and acid phosphatase (EC 3.1.3.2), inter alia. Investigations have been reported using DP to form conjugates of the anti-tumor agent Ara-C (1-.beta.-arabinofuranosylcytosine), with other active agents (steroids), and with lipophilic groups such as aliphatic chains (e.g., alkyl, alkoxyalkyl, etc.). See, e.g., Hong, C. I., Nechaev, A., West, C. R., Biochem. Biophys. Res. Comm., 94, (1980) 1169; Ryu, E. K., Ross, R. J., Matsushita, T., MacCoss, M., Hong, C. I., West, C. R., J. Med. Chem., 25, (1982) 1322; Hong, C. I., Kirisits, A. J., Nechaev, A., Buchheit, D. J., West, C. R., J. Med. Chem., 1985) 171; Rosowsky, A., Ross, H. S., Wick, M. M., J. Med. Chem., 25, (1982) 171; and Hong, C. I., S. H., Schliselfeld, L., Buchheit, D. J., Nechaev, A., Kirisits, A. J., West, C. R., J. Med. Chem., 31, (1988) 1793. The conjugate was theorized to protect against enzymatic degradation of the active moiety and/or the lipophilic groups had been theorized to enhance cellular uptake of the agent by increasing permeability to cell membranes. These investigators, however, report widely varying effects on the bioavailability of the active agent, both increasing and decreasing efficacy, depending on the nature of the lipophilic moiety as well as the nature of the cleavable group, i.e., diphosphate or monophosphate.
Cleavable diphosphate linking groups have never been employed for the purpose of targeting agents to desired, e.g., in vivo, sites. Thus, for example, they have not heretofore been linked to polypeptide, e.g., proteinaceous, or other targeting groups. In part, this is due to the difficulty of synthesis and of appropriately attaching the necessary portions to each other while retaining all attributes necessary for a successful site-directing conjugate. For example, direct specific phosphorylation of a hydroxy or other nucleophilic group of an antibody is not viable, since there would be no selectivity towards, e.g., OH or NH.sub.2 group. Amino or hydroxy groups, respectively, and other nucleophilic groups would also be phosphorylated. Moreover, a linker comprising such a phosphorylated group would not cleave as desired. As a result, a synthetic linker needs to be designed, which is not straightforward. For example, simple straight-chain hydroxy acids are not applicable as linking components due to spontaneous lactone formation, and glyceric acid with isopropylidene protected hydroxy groups is also inappropriate, since self-destructive deketalization is catalyzed by internal carboxylic acids.